||Activated chromatin Euchromatin
||Nonactivated chromatin, Facultative and obligate heterochromatin, euchromatin
||Less condensed, unfolding of functional domains (2040 kbp) exhibit
|DNA methylation (CG sites)
||DNase-I-sensitive sites mv-sites unmethylated
||DNase I resistant
||H1-deprivated; core histones highly acetylated
||H1-enriched; association with special H1 isofores, e.g., H5; H2A/H2B underacetylated; eventually H2A modified by ubiquitin
||Presence of RNAP and RNP depends on the actual transcription state
|TABLE 1. Properties of chromatin
- Saline citrate (1/10 SSC)
- 1.0 N H2SO4
- Refrigerated preparative centrifuge
- Absolute ethanol
- 0.01 M sodium phosphate buffer, pH 7.0 + 1% (w/v) SDS + 0.1% (v/v)
- 10% acrylamide gels (10%T:5%C) with 0.1% (w/v) SDS
- 7% (w/v) acetic acid
- 0.25% Coomassie Blue
Dissolve crude chromatin in cold dilute saline citrate (0.015 M NaCl + 0.001 M
sodium citrate) to a final DNA concentration of 500 µg/mL.
- Stir the solution on ice and slowly add ¼ volume of cold 1.0 N H2SO4.
Continue stirring for 30 minutes.
- Centrifuge the suspension at 12000 xg for 20 minutes at 4°C. Save the
supernatant. For maximum yield, break up the pellet, resuspend in fresh,
cold 0.4 N H2SO4, re-extract, centrifuge, and add the resulting supernatant
to the first.
- Add 4 volumes of cold absolute ethanol to the supernatant and store for
24 hours at –10° C to precipitate the histone-sulfates.
- Collect the precipitate by centrifugation at 2000 xg for 30 minutes.
- Decant as much of the alcohol as possible, and resuspend the pellet in
cold absolute ethanol.
- Centrifuge at 10000 xg for 15 minutes.
- Collect the pellet and freeze dry for later analysis.
To continue with the electrophoresis, carefully weigh the histone protein
sample and dissolve in 0.01 M sodium phosphate buffer with a pH 7.0
and containing 1% sodium dodecyl sulfate and 0.1% β-mercaptoethanol;
the final volume should contain approximately 300 mg of protein in 100 mL
- Prepare the electrophoresis chamber with a 10% acrylamide gel with 0.1%
- Add separately 25 µL of the dissolved protein and 25 µL of protein standards
50 µL of 0.1% SDS, 0.1% β-mercaptoethanol in buffer
5 µL of β-mercaptoethanol
1 µL of 0.1% bromophenol Blue in water
- Mix thoroughly and apply the histone extract and protein standards to
separate wells of the electrophoresis gel.
- Separate the proteins in the anode direction (anionic system).
- The addition of SDS anions to the proteins results in negatively charged
proteins, which will separate according to molecular weight.
- Electrophoresis is carried out in the standard manner. The buffer utilized
- 0.025 M Tris-0.192 glycine and 0.1% SDS, pH 8.3.
- Proteins are separated by a current of 3–4 mA per gel until the
bromophenol marker reaches the bottom of the tube (about 7 hours at
3 mA, and 4 hours at 4 mA).
- Stain the gels with 0.25% Coomassie Blue for 2 hours.
- Destain and store in 7% acetic acid.
- Scan the gels and determine the molecular weights of each component.
Preparation of a total histone fraction from nuclei is normally accomplished by
extraction with a dilute acid or a high-molarity salt solution. The acidic extraction
removes histones from DNA and nonhistones immediately, while the dissociation
of chromatin in salt solutions will require further purification. In either event,
the histones themselves are subdivided into 5 major types, designated as H1,
H2, H3, H4, and H5. H2 dissociates into 2 peptides, which are thus designated
as H2A and H2B. The classification of histones is based on their electrophoretic
Nonhistone proteins can also be extracted and separated by electrophoresis.
Whereas histones have only 5 major types, nonhistones are extremely
heterogeneous and up to 500 different proteins have been identified from one
cell type, while the major proteins comprise less than 20 types.
The extraction of chromatin DNA was possible with the 7 M urea-3 M
NaCl extraction. Further analysis of DNA will be undertaken as part of a later
lab exercise (on transcription), and the DNA sample from this lab may be kept
lyophilized and frozen until that time.
For our current needs, it is sufficient to note that the genes are composed
of DNA, and that various specific regions of the DNA/genetic information can
be physically isolated to a specific locus on a chromosome. This, in turn, is
readily observed and correlated with banding patterns, such as those in the
fruit fly polytene chromosomes.